JP4925540B2 - 5-helix protein - Google Patents

Abstract

Five-Helix protein, which comprises the three N-helices and at least two, but not three, of the three C-helices of the trimer-of-hairpin structure of HIV gp41, separated by linkers, such as amino acid residue linkers, is disclosed. Six-Helix protein, which includes the three N-helices and the three C-helices of the trimer-of-hairpin structure of HIV gp41, separated by linkers, is also disclosed.

Description

（配列番号：１）
【００２８】
アミノ酸の一文字表記により、６−ヘリックスのアミノ酸配列を以下に示す：

（配列番号：２）
【００２９】
５−ヘリックスタンパク質を、種々の方法で作製することができる。例えば、実施例１に記載のように、酵素（トリプシン）消化によってより大きなタンパク質（６−ヘリックスなど）から作製することができる。あるいは、公知の方法および発現系を使用して、全５−ヘリックスタンパク質をコードする１つのＤＮＡまたは２つもしくはそれ以上のＤＮＡ「単位」（それぞれＮ−ヘリックスタンパク質の一部（例えば、１つまたは複数のＮ−ヘリックス、１つまたは複数のＣ−ヘリックス）をコードする）であり得る５−ヘリックスタンパク質コードＤＮＡの発現によって作製することができる。大腸菌などの適切な宿主細胞における５−ヘリックス遺伝子の直接発現によって５−ヘリックスの発現率および精製率を有意に改善することができる。このアプローチでは、５−ヘリックス遺伝子は、最終的な５−ヘリックスタンパク質中に存在する残基をコードする。Ｃ−末端Ｈｉｓタグを結合して精製を容易にすることができる（その後タグを除去するプロテアーゼ開裂部位を有するまたは有しない）。次いで、６−ヘリックスから出発する５−ヘリックスの作製に必要なタンパク質分解を生ずる開裂工程および折りたたみ工程を用いないで直接タンパク質を使用することができる。この５−ヘリックス分子を、折りたたまれた活性分子として発現させることができ、これにより生物学的選択またはスクリーニングで使用してその性質を至適化することができる。あるいは、タンパク質合成法を使用して、５−ヘリックスタンパク質を作製することができる。５−ヘリックスの５つのヘリックスを、（少なくとも１つ（１つまたは複数）のアミノ酸残基のリンカー手段または他の手段などによって）共有結合させて、生理学的条件下で安定で、５−ヘリックスの残りの表面がＣ３４ペプチドの結合に利用可能なように存在するように正確に折りたたまれたタンパク質を形成することができる。３つのＮ−ヘリックスおよび２つを超える（しかし、３つの完全体未満）のＣ−ヘリックスが存在する実施形態では、ヘリックスを同様に結合することができる。
【００３０】
５−ヘリックスは、Ｎ−およびＣ−ヘリックス領域の両方ならびにリンカー成分に広範な種々の配列を有することができ、これは、Ｌ−アミノ酸残基、Ｄ−アミノ酸残基、またはＬ−およびＤ−アミノ酸残基の組み合わせで構成可能である。アミノ酸残基を改変することができる。５−ヘリックスは、ヘリックスおよびリンカーの残基に加えてアミノ酸残基を含み得る（例えば、分子の安定化のため）。本明細書中に記載の５−ヘリックスを安定性および活性を向上させるように変化させることができるようである。Ｎ−およびＣ−ヘリックスに連結させるループのデザイン（長さおよび組成の両方）の小さな変化およびＮ−およびＣ−ヘリックスの正確な境界は、５−ヘリックスの安定性、収量、および活性に有意な効果を有するようである。
【００３１】
現在構築されているものと同様に、５−ヘリックスは、Ｎ−ヘリックス中心に沿って４０個のアミノ酸が取り囲んでいるＣ−ペプチド結合部位を暴露している。５−ヘリックス（あるいは関連分子）上のＣ−ペプチド結合部位のより短いセグメントを暴露するストラテジーには、より長い暴露したエピトープへの短いＣ−ペプチド配列を結合することを含む。この型の分子は、Ｎ−ヘリックス中心に沿ったより短いエピトープに対して特異的に標的化された薬物の開発の一助となり得る。例えば、１つのポケット領域（ＩＱＮ１７に見られるものと類似；Ｄ．Ｍ．Ｅｃｋｅｒｔら、Ｃｅｌｌ、９９、１０３〜１１５（１９９９））を、そこ（Ｃ３４の最初の約１０残基程度）に結合する残基を欠くＣ−ペプチドに結合することによって５−ヘリックス中に暴露されうる。これらの短いＣ−ペプチド配列は、共有結合の架橋または暴露したエピトープの一部を被覆するために５−ヘリックス配列を単に伸長することを含む種々の手段によって５−ヘリックスに結合することができる。
【００３２】
５−ヘリックスは種々の状況で有用である。本明細書中に記載のように、５−ヘリックスは、ウイルス膜融合の強力なインヒビターであるので、ウイルスがＨＩＶ感染細胞に作用する細胞に侵入する前に（現在の実用的治療とは異なる）ウイルスに作用する。５−ヘリックスは可溶性であり、本明細書中に記載の条件下で安定であることが認められた。腎臓でのクリアランス速度を減少させるために分子量を増加させた５−ヘリックス変異型を作製する（オリゴマー形成または巨大タンパク質を結束することによる）ことも可能なはずである。さらに、５−ヘリックス二量体をジスルフィド架橋によって作製して、５−ヘリックス「単量体」よりも小さな範囲に濾過した分子を作製することができる。したがって、Ｃ−ペプチドのものと比較した場合、二量体は生物学的利用能が向上していると予想するのが妥当である。
【００３３】
５−ヘリックスは、ウイルス侵入後のウイルスタンパク質を標的とする標準的な治療と異なり、ウイルスの細胞への侵入を防止するので、５−ヘリックスを予防的に使用して感染を予防するか感染を生じる範囲を減少させることができる。このような治療の１つの用途は、病院などで起こり得るニードルスティックによる損傷またはＨＩＶで汚染した針を共有する状況である。例えば、細胞へのＨＩＶ侵入を予防または減少させるために針を刺し且つＨＩＶに感染しているか感染しているかもしれない個体に十分量の５−ヘリックス（治療有効量）を１回または複数回で投与することができる。５−ヘリックスを、例えば、静脈内注射または筋肉内注射によって投与することができる。
【００３４】
本発明の１つの実施形態では、５−ヘリックスを使用して、個体のＨＩＶ感染を減少させる。この実施形態では、個体の細胞のＨＩＶ感染を（全体的または部分的に）減少させるのに十分な量で、５−ヘリックス自体または適切な宿主細胞またはベクターでの５−ヘリックスコードＤＮＡの発現を介してのいずれかによって５−ヘリックスを個体に投与する。すなわち、ＨＩＶ感染を減少するのに十分な用量（有効用量）の５−ヘリックスを、細胞へのＨＩＶ侵入を（全体的または部分的に）阻害する様式（例えば、注射、局所投与、静脈内経路）で投与する。１つの実施形態では、個体への５−ヘリックスタンパク質を発現する細胞の導入により遺伝子治療アプローチを使用して有効用量を得る。５−ヘリックスを、さらなる感染を減少させるためにＨＩＶ感染した個体に投与するか、感染を予防するか感染が生じる範囲を減少させるために感染していない個体に投与することができる。
【００３５】
適切な担体（例えば、生理学的に許容されうる緩衝液）中に５−ヘリックスを含む医薬組成物は、本発明の主題である。医薬組成物は、予防および治療目的に有用であり、種々の経路（例えば、注射、局所投与、静脈内経路）を介して投与することができる。
【００３６】
５−ヘリックスは、１つのインタクトなＣ−ヘリックス結合部位を示すので、膜融合を阻害する薬物のスクリーニングに有用である。５−ヘリックスは、ＩＱＮ１７が暴露するよりもより大きく且つ強固な潜在的薬物のスクリーニング標的を暴露している。この分子６−ヘリックスおよび５−ヘリックス（Ｄ４）は、これらの研究における有用なネガティブ対照である。
【００３７】
５−ヘリックス暴露エピトープを、抗体（特に、公知の方法を使用した中和抗体）産生用の抗原として使用することができる。抗体は、モノクローナルまたはポリクローナルであり得る。
【００３８】
５−ヘリックスの血清安定性を、その治療可能性を確認するために公知の方法を用いて試験することができる。５−ヘリックスが分解している場合、最も有望な攻撃／分解部位は、グリシン／セリンリンカー領域である。この場合、異なるリンカー領域を分解して試験することができる（以下を参照のこと）。これらの抗５−ヘリックスの血清および腹水の阻害能力を、標準的な融合アッセイを使用して試験することができる。
【００３９】
５−ヘリックスの外側の表面は例えば、生物学的利用能を向上させ、毒性を減少させ、免疫クリアランスを回避するために変化させることができる。５−ヘリックスは強力な阻害活性を示すが６−ヘリックスの束は示さないので、これは阻害を担うポケット領域を含む暴露した溝である。残りの分子は、単に暴露した溝を表示するための足場を提供している。したがって、この足場を、５−ヘリックスの阻害活性に有害な影響を与えること無く改変することができる。足場の改変により、いくつかの利点を得ることができる。第１に、５−ヘリックスの複数回投与が必要な手順を容易にする。例えば、抗ＨＩＶ治療薬として５−ヘリックスを使用する場合、複数回投与が必要であり得る。投与の延長後、個体は身体からのそのクリアランスを増大させると考えられる５−ヘリックスに対する抗体を産生し得る。複数のバージョンの５−ヘリックスの有用性は、既存の抗体を避けることにより、この問題を回避することを助ける。第２に、例えば、足場があまり免疫原性を示さない外側の表面上のグリコシル化部位の導入によって５−ヘリックスのバージョンを設計可能であり得る。ワクチン研究用に、この改変は、足場とは対照的に暴露した溝に免疫応答を偏向させる一助となる。
【００４０】
ｇｐ４１のＣ−ヘリックス領域の結合によりＨＩＶ感染が防止されるという所見により、ＨＩＶワクチンを構築するためのストラテジーが示唆される。Ｃ−ペプチドによるＨＩＶ阻害アナログである５−ヘリックスは、前ヘアピン中間体と呼ばれるｇｐ４１の融合中間体への結合によってｇｐ４１を阻害するようである。Ｃ−ペプチドインヒビターはこの中間体のＮ−ペプチド領域に結合することによって機能する一方で、５−ヘリックスはＣ−ペプチド領域に結合することによって機能するようである。これらの検討材料により、ｇｐ４１のＣ−ペプチド領域がＨＩＶ侵入インヒビター開発用の良好な薬物標的であることが強く示唆される。さらに、中和抗体を惹起させるための免疫原としてＣ−ペプチドベースの構築物を使用することが可能である。５−ヘリックスの場合、阻害の標的は、Ｃ−ペプチド領域のヘリックス高次構造であるが、Ｃ−ペプチド領域の他の高次構造を標的する試薬もまた阻害活性を示し得る。
【００４１】
最近のワクチン研究（Ｒ．Ａ．ＬａＣａｓｓｅら、Ｓｃｉｅｎｃｅ，２８３、３５７〜３６２（１９９９））は、エンベロープ媒介融合過程の中間体が中和抗体を強く惹起することができることを示唆している。このような融合中間体に対する抗体は、エンベロープタンパク質の保存領域を標的化し、それゆえ、広範な範囲のウイルス株を中和するようである。Ｃ−ペプチド領域に対する抗体は、高度に保存され且つ融合過程に重要な領域を標的化する。
【００４２】
ヘアピン三量体は、多数のウイルス膜融合タンパク質の共通の特徴である。これは、インフルエンザ、エボラ、ＳＶ５（シミアンパラインフルエンザウイルス５）、およびＲＳＶ（ヒト呼吸器合胞体ウイルス）の結晶構造で認められている。さらに、レトロウイルス、パラミクソウイルス、およびフィロウイルスファミリーの多数の他のメンバーが、このモチーフを含むと予想されている。関連する脊椎動物小胞融合タンパク質に類似の構造が認められている。本明細書中に記載の基本的ストラテジーを、融合を阻害するためにこれらの任意の系に適用することができる。本発明の１つの主題は、ウイルスとウイルスエンベロープタンパク質（例えば、ウイルスエンベロープタンパク質のＣ−ペプチド領域）に結合してエンベロープ化(enveloped) タンパク質のヘパリン三量体形成を阻害する薬物との接触によるエンベロープを有するウイルス（ウイルスエンベロープタンパク質を含むウイルス）のヘアピン三量体の形成阻害方法である。
【００４３】
本発明を、以下の実施例によって例示するが、これらは決して本発明の制限を意図しない。
【００４４】
実施例１：５−ヘリックスの産生
５−ヘリックスのデザインは、Ｎ３６／Ｃ３４ ６−ヘリックス束状構造に基づいた（Ｄ．Ｃ．Ｃｈａｎら、Ｃｅｌｌ、８９、２６３〜２７３、１９９７）。５−ヘリックスのために、各ペプチド領域をそのＮ末端上の３つの残基およびそのＣ末端上の１つの残基によって伸長し（Ｎ３６およびＣ３４と比較）、最終的なＮ４０およびＣ３８セグメントを作製した（それぞれＨＩＶ−１ ＨＸＢ２ ｇｐ１６０の代表的な残基５４３〜５８２および６２５〜６６２）。Ｎ４０の後ろに−ＧＧＳＧＧ−リンカー、Ｃ３８の後ろに−ＧＳＳＧＧ−リンカーを使用して３つのＮ４０および２つのＣ３８セグメントを連結させた。全ての構築物は、翻訳開始用のＮ末端Ｍｅｔを含む。Ｃ末端のみが異なる以下の２つの異なる５−ヘリックスタンパク質をこの研究のために作製した：（ｉ）−ＧＧ（Ｈ）６で終わるＨｉｓタグ化５−ヘリックスおよび（ｉｉ）−ＧＧＲで終わる非タグ化５−ヘリックス。さらに、トリプシン切断リンカー（−ＧＧＲ−）によって５−ヘリックス骨格をＨｉｓタグ化Ｃ−ペプチド（Ｃ３７−Ｈ６）（実施例４を参照のこと）に連結した６−ヘリックスと呼ばれる第３の構築物を作製した（図１０および１１Ａ〜１１Ｃを参照のこと）。
【００４５】
全てのＤＮＡ構築物を、大腸菌ＸＬ１−Ｂｌｕｅ（ｒｅｃＡ株、Ｓｔｒａｔａｇｅｎｅ）を使用してｐＡＥＤ４ベクター［Ｄ．Ｓ．Ｄｏｅｒｉｎｇ、Ｐ．Ｍａｔｓｕｄａｉｒａ、Ｂｉｏｃｈｅｍｉｓｔｒｙ、３５、１２６７７〜１２６８５、１９９６］に連続的にクローン化したＰＣＲカセットから構築した。全てのタンパク質を、ＩＰＴＧ（０．４Ｍ）で３時間誘導する前に２×ＹＴ中で０．５〜０．７のＯＤ（５９０ｎｍ）まで増殖させた大腸菌ＲＰ３０９８株で組換え発現させた。細菌ペレットを、完全無ＥＤＴＡプロテアーゼインヒビタータブレット（Ｒｏｃｈｅ）を補足したＴｒｉｓ／ＮａＣｌ緩衝液（Ｑｉａｅｘｐｒｅｓｓｉｏｎｉｓｔ ｂｏｏｋｌｅｔ、１９９９年３月、Ｑｉａｇｅｎ）に再懸濁し、その後精製する日まで−２０℃で凍結した。解凍した再懸濁物を溶解し（超音波処理またはフレンチプレス）、遠心分離して（３５，０００×ｇで３０分間）、封入体から可溶性画分を分離した。
【００４６】
Ｈｉｓタグ化５−ヘリックス（プラスミドｐ−５ＨｅｌｉｘＨ６から作製）を、８Ｍ尿素のＴＢＳ（５０ｍＭのＴｒｉｓ（ｐＨ８．０）、１００ｍＭのＮａＣｌ）および１０ｍＭイミダゾール溶液に再懸濁した封入体から直接精製した。混合物を遠心分離（３５，０００×ｇで３０分間）によって明澄化後、室温でＮｉ−ＮＴＡアガロース（Ｑｉａｇｅｎ）カラムに結合させた。４０ｍｌ（約５カラム体積）の６Ｍ尿素／ＴＢＳ／１００ｍＭイミダゾール溶液でタンパク質を溶出した。室温での１リットルの２０ｍＭのＴｒｉｓ（ｐＨ８．０）撹拌溶液の滴下によってタンパク質の再折り畳みを行った。次いで、再折りたたみタンパク質を、Ｎｉ−ＮＴＡアガロースカラムの通過によって再濃縮し、２０ｍｌ（約２カラム体積）の１００ｍＭイミダゾールのＴＢＳ溶液で溶出した。
【００４７】
非タグ化５−ヘリックスを６−ヘリックスのタンパク質分解によって産生し（以下を参照のこと）、５−ヘリックス／Ｃ３７−Ｈ６複合体を作製した。トリプシン消化後（ＴＢＳ中に１：２００の重量比、室温で１時間、Ｓｉｇｍａ）、５−ヘリックス／Ｃ３７−Ｈ６複合体をＮｉ−ＮＴＡアガロースに結合させ、大規模に洗浄して過剰なトリプシンを除去した。ビーズを８ＭのＧｕＨＣｌ／ＴＢＳ中に再懸濁し、加熱（７０℃）して複合体を変性させた。変性５−ヘリックスを含む非結合画分を、８Ｍ尿素／２０ｍＭのＴｒｉｓ（ｐＨ８．０）（室温で４時間）および４Ｍ尿素／２０ｍＭのＴｒｉｓ（ｐＨ８．０）（４℃で一晩）で連続的に透析した。タンパク質をＤＥＡＥカラム（Ｆａｓｔｆｌｏｗ、Ｐｈａｒｍａｃｉａ）にロードし、室温で４時間２０カラム体積で逆尿素勾配（４Ｍ〜０Ｍ尿素の２０ｍＭのＴｒｉｓ溶液（ｐＨ８．０））で運転した。ＮａＣｌ勾配（０〜３００ｍＭ）の２０ｍＭのＴｒｉｓ溶液（ｐＨ８．０）（１０カラム体積）を使用してタンパク質をＤＥＡＥ樹脂から溶出した。６−ヘリックス（プラスミドｐ−６へリックスから作製）を、細菌溶解物の可溶性画分から直接精製した。溶液をＮｉ−ＮＴＡアガロースカラムに通し、１０カラム体積のイミダゾール勾配（１０〜２５０ｍＭ）のＴＢＳ溶液で溶出した。
【００４８】
すべてのタンパク質について、ＴＢＳ中でのゲル濾過（Ｓｅｐｈａｃｒｙｌ Ｓ２００ＨＲまたはＳｕｐｅｒｄｅｘ７５）によって凝集物から単量体を分離した。ＳＤＳ−ＰＡＧＥで判断したところタンパク質は９５％を超える純度であり、少なくとも３ｍｇ／ｍｌに濃縮することができる。すべてのペプチドおよびタンパク質の濃度を、６ＭＧｕＨＣｌ中での２８０ｎｍでの吸光度によって同定した［Ｈ．Ｅｄｉｌｈｏｃｈ，Ｂｉｏｃｈｅｍｉｓｔｒｙ６、１９４８〜１９５４、（１９６７）。］
【００４９】
実施例２：５−ヘリックス／Ｃ−ペプチド相互作用の特異性および膜融合の５−ヘリックスによる阻害の評価
５−ヘリックス／Ｃ−ペプチド相互作用の特異性を、Ｈｉｓタグ化Ｃ−ペプチド（大腸菌中で独立して発現し、逆相ＨＰＬＣで精製したＣ３７−Ｈ６）およびＮｉ−アガロース沈殿を使用して試験した。３０μＭのＣ３７−Ｈ６を含むＴＢＳ中で、Ｎｉ−アガロースによって１６μＭの５−ヘリックスが完全に沈殿する。１５０μＭのＣ３４（Ｈｉｓタグを含まない、化学合成し、ＨＰＬＣで精製）の添加により、５−ヘリックスの沈殿量が実質的に減少する。Ｃ３７−Ｈ６とＣ３４との有効な競争は、５−ヘリックスが特異的様式でＣ−ペプチドに結合することを示す。ＣＤ実験および競合結合アッセイにより、５−ヘリックスが予想された高次構造に折りたたまれていることが示唆される。すなわち、これらの結果は、５−ヘリックスが暴露したＣ−ペプチド結合部位を含むという予想を支持するものである。
【００５０】
５−ヘリックスがｇｐ４１のＣ領域と相互作用して融合タンパク質の融合を阻害する能力を評価するアッセイを行った。５−ヘリックスおよび６−ヘリックスによるこの膜融合の阻害を、細胞ベースアッセイを使用して評価した。タンパク質５−ヘリックスおよび６−ヘリックスを、５％ＦＣＳを含む改変ＤＭＥＭ培地で連続希釈し、スライドチャンバーに等分した。Ｔａｔプロモーターの調節下でＣＤ４および補助受容体を発現し、βガラクトシダーゼ遺伝子を含むＨＥＬＡ細胞（４×１０⁴ ）を添加する。ｇｐ１６０（ｇｐ１２０／ｇｐ４１の前駆体タンパク質）およびＴａｔを発現するＣＨＯ細胞（２×１０⁴ ）も添加する。４００μｌのミニ培地を３７℃で８〜２４時間インキュベートし、融合細胞（シンシチウム）はβガラクトシダーゼを転写および翻訳する。グルタルアルデヒド中に細胞を固定し、Ｘ−ｇａｌ／Ｆｅ溶液に１時間暴露する。βガラクトシダーゼを含むシンシチウムは、青緑色に変色する。このアッセイでは、５−ヘリックスは、シンシチウム形成の強力な阻害を示している（ＩＣ₅₀は１０〜２０ｎＭ；あるアッセイでは１３ｎＭのＩＣ₅₀であった）。６−ヘリックスは１μＭの濃度でさえ感知できるほどには融合を妨害しない。
【００５１】
阻害剤としての５−ヘリックス暴露エピトープの特異性を評価し、汚染物質を除外するために、Ｃ−ペプチドとの混合実験を行った。濃度２００ｎＭの５−ヘリックスを、１００、１６６、１９０、および２１０ｎＭのＣ３４と混合する。使用濃度では、遊離の５−ヘリックスおよび遊離のＣ３４は、ミニ培養物中のほとんど全てのシンシチウムを阻害するはずである。Ｃ３４が５−ヘリックスに対して過剰にある（すなわち、２１０ｎＭ）５−ヘリックス／Ｃ３４混合物ではシンシチウム形成は妨害されるが、Ｃ３４濃度が５−ヘリックス濃度より低い５−ヘリックス／Ｃ３４混合物におけるシンシチウム形成は部分的に妨害されない（図３Ｂ）。それに対して、６−ヘリックスの存在下ではＣ３４は全てのシンシチウム形成をブロックする。
【００５２】
５−ヘリックスおよび６−ヘリックスの阻害可能性を、ウイルス融合実験で再現した。ルシフェラーゼレポーター遺伝子を含むように改変したＨＩＶを、希釈タンパク質の存在下、３７℃で６時間ＣＤ３４および補助受容体を発現するヒト骨肉種（ＨＯＳ）細胞と混合する。ウイルス溶液を交換し、ＨＯＳ培養物を新鮮な培地中で４８時間以上インキュベートする。ルシフェラーゼ活性はルミノメーターで測定する。このアッセイでは、５−ヘリックスは１０ｎＭ未満のＩＣ₅₀でルシフェラーゼ活性を阻害する。また、６−ヘリックスは、１μＭまで感知できるほどの妨害を示さない（図３Ａおよび３Ｃ）。
【００５３】
実施例３：５−ヘリックス（Ｄ４）のデザインおよび評価
５−ヘリックス（Ｄ４）では、Ｈｉｓタグ化５−ヘリックスのＣ−ペプチド結合部位中の４つの非常に保存された残基（Ｖａｌ５４９、Ｌｅｕ５５６、Ｇｌｎ５６３、およびＶａｌ５７０）を、最後の（第３の）Ｎ４０セグメント中でＡｓｐに変異させた。構築物［ｐ−５Ｈｅｌｉｘ（Ｄ４）］を組換え発現し、Ｈｉｓタグ化５−ヘリックスと同一の様式で精製した。Ｈｉｓタグ化５−ヘリックスおよび５−ヘリックス（Ｄ４）タンパク質は、同一の楕円率を有し：（共に、４℃のＴＢＳ中で［θ］₂₂₂ ＝−２８，１００±１５００ｄｅｇ ｃｍ² ｄｍｏｌ^-1（約１００％の推定ヘリックス含有率））、両タンパク質は、ＴＢＳ中での熱変性（９８℃を超えるＴｍ）および２５℃でのＧｕＨＣｌ化学変性（Ｃｍ値は５−ヘリックス（Ｄ４）で約６Ｍ、Ｈｉｓタグ化５−ヘリックスで約７．２Ｍ）に対して非常に安定である。５−ヘリックス（Ｄ４）の安定性がわずかに減少したのは、この状況で５−ヘリックス表面上の大部分が疎水性の溝（groove）の中に存在するＡｓｐ残基のヘリックス傾向の低さおよび電荷に影響を反映しているようである。
【００５４】
実施例４：Ｈｉｓタグ化Ｃ−ペプチドＣ３７−Ｈ６
ペプチドＣ３７−Ｈ６は、以下の配列のＨｉｓタグ化Ｃペプチドである：
ＧＧＨＴＴＷＭＥＷＤＲＥＩＮＮＹＴＳＬＩＨＳＬＩＥＥＳＱＮＱＱＥＫＮＥＱＥＬＬＧＨＨＨＨＨＨ（配列番号５）。このペプチドはＨＩＶ−１ ＨＸＢ２残基６２５〜６６１（下線）に由来し、全Ｃ３４配列（Ｗ６２８〜Ｌ６６１）を含む。Ｃ３７−Ｈ６を−ＧＧＲ−リンカーでＣ３７−Ｈ６に結合された１つのＮ４０セグメントからなる組換え発現構築物ｐ４−ＮＣ１．１のトリプシン消化から作製する。発現後、ＮＣ１．１を、６−ヘリックスと同一の様式で細菌溶解物の可溶性画分から精製する。トリプシン消化（非タグ化５−ヘリックスと同一の条件）によりＣ３７−Ｈ６が得られ、次いで、Ｖｙｄａｃ Ｃ−１８カラムおよび直線勾配の、０．１％トリフルオロ酢酸を含むアセトニトリル水溶液を使用した逆相ＨＰＬＣによって均一に精製する。Ｃ３７−Ｈ６の同一性を、質量分析（ＭＡＬＤＩ−ＴＯＦ、ＰｅｒＳｅｐｔｉｖｅ）によって確認した。Ｃ３４と同様に、Ｃ３７−Ｈ６は、ＨＩＶ−１膜融合の強力なインヒビターである（細胞−細胞融合アッセイにおいてＩＣ₅₀は約１ｎＭ）。
【００５５】
図２Ａ〜２Ｄのデータを、５−ヘリックスの非タグ化変形形態を使用して作製したが、Ｈｉｓタグ化変形形態と類似の結果が得られた（実施例３を参照のこと）。特記しない限り、ＴＢＳ緩衝液中でＣＤ（Ａｖｉｖ６２ＤＳ）実験を行った。図２Ｂでは、タンパク質濃度は、ＴＢＳサンプルについては１ｍＭであり、ＧｕＨＣｌ／ＴＢＳサンプルについては０．５４ｍＭであった。図２Ｄでは、１ｍｌチャンバー（４．３７５ｍｍ／チャンバー光路長）を有する石英混合セル（Ｈｅｌｍａ）を使用した。混合前の２０ｍＭのＴｒｉｓ（ｐＨ８．０）／２５０ｍＭのＮａＣｌ中のポリペプチド濃度は５．９ｍＭの（５−ヘリックス）および６ｍＭの（Ｃ３７−Ｈ６）であった。
【００５６】
１６ｍＭ非タグ化５−ヘリックス、３０ｍＭのＨｉｓタグ化Ｃ３７−Ｈ６、および／または１５０ｍＭのＣ３４を含む２０ｍｌのＴＢＳ中で５−ヘリックス沈殿実験（図２Ｃ）を行った。Ｎｉ−ＮＴＡに溶液を添加し、室温で１０分間インキュベートした。非結合上清を除去後、１ｍｌのＴＢＳでビーズを２回洗浄し、５００ｍＭのイミダゾールで溶出した。Ｎｉ結合および非結合サンプルを、１６．５％Ｔｒｉｓ−トリシンポリアクリルアミドゲル（Ｂｉｏｒａｄ）上で泳動し、Ｇｅｌ−ｃｏｄｅ Ｂｌｕｅ（Ｐｉｅｒｃｅ）で染色した。
【００５７】
実施例５：Ｈｉｓタグ化５−ヘリックス
図３Ａ〜３Ｃの全てのデータを、Ｈｉｓタグ化５−ヘリックスを使用して作製した（実施例１を参照のこと）。細胞−細胞融合アッセイ（図３Ｂ）を記載のように行った（Ｄ．Ｃ．Ｃｈａｎら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ９５、１５６１３〜１５６１７、１９９８）。以前に詳述されたアッセイ（Ｄ．Ｃ．Ｃｈａｎら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ、９５、１５６１３〜１５６１７、１９９８）を少し改変した組換えルシフェラーゼレポーターアッセイを使用してウイルス感染阻害を研究した。簡単に述べれば、エンベロープ欠失ＨＩＶ−１ゲノムＮＬ４３ＬｕｃＲ^- Ｅ^- ［Ｂ．Ｋ．Ｃｈｅｎら、Ｊ．Ｖｉｏｌ．、６８、６５４〜６６０、１９９４］および以下の４つのｇｐ１６０発現ベクターのうちの１つ：ｐＣＭＶ−ＨＸＢ２（Ｄ．Ｃ．Ｃｈａｎら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ、９５、１５６１３〜１５６１７、１９９８）、ｐＥＢＢ−ＪＲＦＬ（Ｂ．Ｋ．Ｃｈｅｎから提供）、ｐＳＶＩＩＩ−ＵＧ０２４．２、およびｐＳＶＩＩＩ−ＲＷ０２０．５で共トランスフェクトした２９３Ｔ細胞から偽型ウイルスを作製した。プラスミドｐＳＶＩＩＩ−ＵＧ０２４．２およびｐＳＶＩＩＩ−ＲＷ０２０．５を、ＮＩＨ ＡＩＤＳ試薬プログラム（Ｆ．Ｇａｏ、Ｂ．Ｈａｈｎ、およびＤＡＩＤＳ、ＮＩＡＩＤ）から獲得し、これは初代ＨＩＶ−１単離物由来のエンベロープタンパク質をコードする。ウイルスを含む上清を以前に記載のように調製し（Ｄ．Ｃ．Ｃｈａｎら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ９５、１５６１３〜１５６１７、１９９８）、これを使用してＨＯＳ−ＣＤ４細胞（ＨＸＢ２およびＵＧ０２４．２）またはＨＯＳ−ＣＤ４−ＣＣＲ５細胞（ＪＲＦＬおよびＲＷ０２０．５）のいずれかを感染させた。ＨＩＨ ＡＩＤＳ試薬プログラム（Ｎ．Ｌａｎｄａｕ）から細胞を得た。図３Ａでは、標準的な２４ウェル形式でウイルス感染性アッセイを行った（Ｄ．Ｃ．Ｃｈａｎら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ９５、１５６１３〜１５６１７、１９９８）。図３Ｃのデータを、以下の９６ウェル形式で行ったアッセイから得た：ウイルス含有上清（１０ｍｌ）および培地（９０ｍｌ）を５０％の密集度のＨＯＳ細胞上に被せた。３７℃で２日間のインキュベーション後、１００ｍｌの溶解緩衝液（ルシフェラーゼアッセイシステム、Ｐｒｏｍｅｇａ）中に細胞を回収し、製造者のプロトコールに従ってその１０ｍｌを分析した。ラングミュア関数［標準化ルシフェラーゼ活性＝１／（１＋［５−ヘリックス］／ＩＣ₅₀）］への５−ヘリックス滴定データの適合によってＩＣ₅₀値を計算した。
【００５８】
本発明を、特に好ましい実施形態を参照して表示および記載しているが、添付の特許請求の範囲に含まれる本発明の範囲を逸脱することなく形態および詳細の種々の変更を行うことができることが当業者に理解される。
【図面の簡単な説明】
【図１】 図１Ａおよび図１Ｂは、ＨＩＶ−１膜融合の標的化を示す。図１Ａは、ｇｐ４１ヘアピン三量体の形成を促進する事象を示すＨＩＶ−１膜融合の略図である。天然の状態では接近不可能なｇｐ４１（赤）のＮ末端融合ペプチドを、ＣＤ４および補助受容体とのｇｐ１２０の相互作用後に標的細胞膜に挿入する（示さず）。前ヘアピン中間体の構造は、Ｎ末端コイルドコイル（灰色）（Ｃ−ペプチド阻害の標的）が暴露している。この一過性構造がヘアピン三量体状態に崩壊して、膜が融合して密接に並行に配置される。図１Ｂは、５−ヘリックス構築物のデザインを示す。リボン図（上）は、ヘアピン三量体の中心構造（左およびＤ．Ｃ．Ｃｈａｎら、Ｃｅｌｌ ８９、２６３〜２７３（１９９７））および５−ヘリックスモデル（右）を示す。内側の灰色のヘリックスはＮ３６ペプチドを示し、外側の青色のヘリックスはＣ３４ペプチドを示す。５−ヘリックスモデルでは１つのＣ−ペプチドが取り除かれており、橙色の線を引いてヘリックス間の接続を示す。５−ヘリックスのデザインでは、短いＧｌｙ／Ｓｅｒペプチド配列（図の下の灰色のバー）によってＮ４０およびＣ３８配列（一文字アミノ酸記号で示す）が交互に結合している（実施例１を参照のこと）。
【図２】 図２Ａ〜図２Ｄは、５−ヘリックスの性質を示す。図２Ａは、２５℃での５−ヘリックス（１０μＭ）の円偏光二色性（ＣＤ）スペクトルである。このスペクトルは、５−ヘリックスタンパク質がデザインから予想される９５％を超えるヘリックス含有率を使用していることを示している。図２Ｂは、ＴＢＳ（黒四角）および３．７Ｍグアニジン（Ｇｕ）ＨＣｌ／ＴＢＳ（白四角）における２２２ｎｍでの楕円率によってモニターした５−ヘリックスの熱変性のグラフである。ＧｕＨＣｌ溶液で認められた変性は、９０％を超えて可逆性である。図２Ｃは、Ｈｉｓタグ化Ｃ−ペプチドでの５−ヘリックスのニッケル（Ｎｉ）−ＮＴＡ沈殿の結果を示す。非タグ化５−ヘリックスおよびＨｉｓタグ化Ｃ−ペプチド（Ｃ３７−Ｈ６と示す）をＮｉ−ＴＮＡアガロースを添加する前に混合し、Ｃ３７−Ｈ６を含む複合体を沈殿させた（レーン１および５ならびに実施例４）。過剰な非タグ化Ｃ−ペプチド（Ｃ３４）の添加により、５−ヘリックス分子が結合画分から非結合画分に変化する（レーン２および６）。図２Ｄは、混合キュベットでの混合前（黒四角）および混合後（白丸）の５−ヘリックスおよびＣ３７−Ｈ６のＣＤスペクトルである。混合による２２２ｎｍでの楕円率の増加は、全体のヘリックス含有率が増加する（会合したＣ−ペプチドあたりのさらなる２８ヘリックス残基に対応する）２種間の相互作用を示す。
【図３】 図３Ａ〜図３Ｃは、実施例２に記載のＨＩＶ−１エンベロープ媒介膜融合の５−ヘリックス阻害の評価結果を示す。図３Ａは、実施例３および５に記載の５−ヘリックス（黒四角）、６−ヘリックス（白三角）、および５−ヘリックス（Ｄ４）（白丸）によるウイルス感染性の滴定評価の結果を示す。データを、２つまたはそれ以上の個別の実験の平均±ＳＥＭで示す。図３Ｂは、５−ヘリックスおよびＣ３４の拮抗阻害活性のグラフである。シンシチウム数を、表示濃度の５−ヘリックス、Ｃ３４、もしくは５−ヘリックスとＣ３４との混合物の存在下または不在下で行った細胞−細胞融合アッセイで測定した。このアッセイでの５−ヘリックスおよびＣ３４のＩＣ₅₀値は、それぞれ１３±３ｎＭおよび０．５５±０．０３ｎＭである（Ｄ．Ｃ．Ｃｈａｎら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ ９５、１５６１３〜１５６１７（１９９８））。データは、対照以外の二連の測定の平均および平均の範囲を示している（５つの測定値の平均±ＳＥＭ）。図３Ｃは、異なるＨＩＶ−１エンベロープ糖タンパク質を含む偽型ウイルスの５−ヘリックス阻害の評価結果を示す。報告したＩＣ₅₀値は、３つの独立した実験の平均±ＳＥＭで示す。
【図４】 図４は、５−ヘリックスとｇｐ４１のＣ−ペプチド領域との相互作用を示すヘリックスのホイール図である。各ヘリックス中のａ〜ｇの位置は、各配列中の４，３−疎水性の７残基反復中の連続した位置を示す。ｇｐ４１ Ｃ−ペプチド中のａおよびｄの位置は、５−ヘリックスのＮ４０コイルドコイル上に暴露したｅおよびｇの位置と相互作用する。ＨＩＶ−１、ＨＩＶ−２、およびＳＩＶ単離物由来の２４７配列（ＨＩＶ−１配列データベース、２０００年８月、ロスアラモス国立研究所）のアラインメントから決定した保存の程度にしたがって残基を囲む：黒四角（＞９０％同一）、灰色の四角（＞９０％保存的置換）、点線の四角（７０〜９０％保存）、囲み無し（＜７０％保存）。図４の作成において、以下のアミノ酸残基群内の置換を保存的置換とみなした： ［Ａｓｐ、Ｇｌｕ］、［Ｌｙｓ、Ａｒｇ］、［Ａｓｎ、Ｇｌｎ］、［Ｐｈｅ、Ｔｙｒ］、［Ｓｅｒ、Ｔｈｒ］、および［Ｖａｌ、Ｉｌｅ、Ｌｅｕ、Ｍｅｔ］。ｇｐ４１のＣ−ペプチド領域のａおよびｄの位置での高い保存度、Ｃ−ペプチド領域の他の位置（特にｃおよびｇ）で著名に欠如している特性は、５−ヘリックスの結合に直接関与しないことに留意のこと。
【図５】 図５は、ＨＩＶ ｇｐ４１の構造の編成である。ヘリックス領域（７残基反復）を灰色で示し、Ｎ−（Ｎ３６）およびＣ−（Ｃ３４、ＤＰ１７８）ペプチドの相対的な位置を示す。ヘリックス領域のリボン図では、Ｎ−ペプチドを淡灰色で示し、Ｃ−ペプチドを濃灰色で示す。
【図６】 図６は、６−ヘリックスおよび５−ヘリックスの配列である。推定ヘリックスセグメントを、配列の積み重ねによって示す。
【図７】 図７は、５−ヘリックスの可能なαヘリックス編成の１つのリボン図である。Ｎ−ヘリックス三量体は淡灰色であり、Ｃ−ヘリックス領域は濃灰色であり、伸長ループ領域は黒色である（Ｄ．Ｃ．Ｃｈａｎら、（Ｃｅｌｌ ８９、２６３〜２７３（１９９７）の構造に基づく）。
【図８】 図８は、細胞−細胞融合アッセイ滴定実験の画像を示す。画像中、シンシチウム（融合細胞を示す）は青色、破片は褐色である。
【図９】 図９は、細胞−細胞融合アッセイ競争実験の画像を示す。漸増量のＣ３４ペプチドと共に２００ｎＭの６−ヘリックスまたは５−ヘリックス中でインキュベートした培養物についてのシンシチウムの量を記録する。
【図１０】 図１０は、５−ヘリックス構築物のデザインを示す図である。この略図は、基本的５−ヘリックス構築物の結合パターンを示す。３つの異なるＣ末端を付加した。６−ヘリックスでは、Ｈｉｓタグ化Ｃ−ペプチドを、５−ヘリックスに結合させて、ヘアピン三量体の完全な６−ヘリックス束を模倣する。Ｎ４０およびＣ３８配列（短いＧｌｙ／Ｓｅｒリンカーを使用して交互に連結した）は、ＨＩＶ ＨＸＢ２ ｇｐ４１のＮ−およびＣ−ペプチド領域に由来する。（赤色および青色で囲んだ残基は、それぞれＮ３６（配列番号：１１）およびＣ３４（配列番号：１２）のペプチド配列を示す。）
【図１１】 図１１Ａ〜図１１Ｃは、本発明のペプチドのアミノ酸配列（配列番号：１〜１０）を示す。[0001]
Related applications
This application is a copy of Michael J. Root, Michael S. Kay, David C.I. Chan and Peter S. Kim (filed Dec. 16, 1999), US Provisional Application 60 / 171,042 entitled “5-Helix Protein” and Michael J. Root, Michael S. Kay, David C.I. Chan and Peter S. Claims the advantages of US provisional application 60 / 234,572 entitled “Protein Design of HIV Invasion Inhibitor” by Kim (filed Sep. 22, 2000). The entire teachings of both referenced provisional applications are incorporated herein by reference.
[0002]
Government aid
This invention was supported in whole or in part by grant number PO1GM56552 from the National Institutes of Health. The United States government has certain rights in this invention.
[0003]
Background of the Invention
HIV is the causative virus of AIDS that is prevalent worldwide. The initial stage of HIV infection includes the fusion of the viral membrane with the target cell membrane and the process of injecting the viral contents into the cell cytoplasm. On the viral side, the molecular complex responsible for the fusion activity includes the surface protein gp120 and the transmembrane protein gp41. It is now believed that gp120 interacts with the protein CD4 and co-receptor on the target cell, changing the higher order structure and inserting the amino terminus (fusion peptide region) of gp41 into the target cell membrane. This structural rearrangement promotes fusion of the virus with the cell membrane, but the mechanism is not well understood.
[0004]
Summary of the Invention
The present invention is folded into a stable structure under the conditions described herein and binds to a peptide corresponding to the C-peptide region of the HIV gp41 protein (referred to as C34) or a portion of this region, such as human cells Relates to a novel protein called 5-helix or 5-helix protein that inhibits HIV infection of mammalian cells. A 5-helix is composed of three N-helices separated by a linker, such as an amino acid residue linker, and three C-helices of at least two but not three trimers of the hairpin structure of HIV gp41. ing. That is, the 5-helix includes at least two of the three N-helices and the three C-helices of HIV gp41. A portion of the third C-helix can also be included, but not all of the third C-helix. Each helix is separated by a linker, preferably an amino acid residue linker, before and after the helix. In one embodiment, the 5-helix can be represented as follows: N-linker-C-linker-N-linker-C-linker-N, where N represents the N-helix and C Represents a C-helix or part of a C-helix). As indicated herein, the term 5-helix or 5-helix protein refers to all such embodiments (although three N-helices and two or more separated by a suitable linker). Containing less than 3 complete C-helices). The amino acid composition of the 5-helix is a structure in which the 5-helix is complementary to the C-peptide region of the HIV gp41 protein, and preferably binds to the peptide of gp41 as a whole or as part of the C34 or C-peptide region of gp41. It can vary greatly as long as the surface is present. That is, the remaining (interactive) surface of the 5-helix (C-peptide binding site, all or part of which is not occupied by C-peptide) is available for binding to the C-peptide region of HIV gp41. Must exist in a form (higher order structure). For 5-helix vaccine and therapeutic applications, the 5-helix must bind (can bind) to the C34 or C-peptide region of HIV gp41. If the 5-helix is used as a drug screening tool or an antibody screening tool, the 5-helix need not (but may not be able to bind) to the C34 or C peptide region of HIV gp41.
[0005]
In one embodiment, the 5-helix has the amino acid sequence of SEQ ID NO: 1. In other embodiments, the 5-helix is structurally complementary to the C-peptide region, preferably there is a surface that binds to the C-34 or C-peptide region, and the addition of at least one amino acid residue, It has an amino acid sequence that differs from SEQ ID NO: 1 by deletion, substitution, or modification. The order of the 5-helix N-helix and C-helix can also be changed, provided that the exposed protein is a higher order structure such that there is a surface complementary to the structure in the C-peptide region of HIV gp41. it can. When the 5-helix protein conformation is retained as described above, the linker can be of any length or composition. The 5-helix can be an L-amino acid protein, a D-amino acid protein, or a combination of L-amino acid residues and D-amino acid residues, which can be modified residues.
[0006]
The present invention further includes a DNA encoding a 5-helix; a method for producing a 5-helix; a method for using the 5-helix (such as a method for inhibiting HIV entry into mammalian cells including human cells) and humans, etc. Methods of eliciting an immune response in an individual of the same; Methods of using DNA encoding a 5-helix, such as in gene therapy; bacteria, humans, and other mammals and other eukaryotic cells that contain and express 5-helix protein-encoding DNA Genetically engineered cells such as cells and methods for using such cells (eg, gene therapy or 5-helix production); compositions such as pharmaceutical compositions containing 5-helixes; 5-helix and HIV envelope proteins (eg, gp120 And a component that binds to a 5-helix complex; including a 5-helix complex Compositions such as pharmaceutical compositions; methods of using such antibodies, such as antibodies that bind to 5-helices, particularly neutralizing antibodies and methods of reducing HIV infection; and inhibiting HIV infection of cells and / or 5-helices The present invention relates to a method for identifying a molecule or compound that binds to a protein.
[0007]
5-helix is an anti-HIV therapeutic agent, prophylactic agent, drug that prevents HIV infection, other anti-HIV therapeutic or prophylactic agent identification (screening) or design reagent, and an antibody that prevents or reduces HIV infection It is useful as an immunogen for inducing In certain embodiments, the invention refers to the compound or molecule to be evaluated as a candidate inhibitor, combining the candidate inhibitor and the 5-helix under conditions suitable for binding of the inhibitor to the 5-helix. Determining if the binding occurs, and if binding occurs, the candidate inhibitor is a compound or molecule that binds to the 5-helix, HIV infection of mammalian cells binding to the 5-helix The present invention relates to a method for identifying a compound or molecule that inhibits the above. The method optionally further comprises determining whether the compound or molecule that binds to the 5-helix inhibits HIV infection of mammalian (eg, human) cells, such as in a cell-based assay. Such a compound or molecule inhibits (in whole or in part) the HIV infection of the cell (eg, by interfering with or interfering with the formation of hairpin trimers).
[0008]
In another embodiment, the invention relates to an individual's HIV comprising introducing a composition comprising a 5-helix and a physiologically acceptable carrier by a suitable route at a dose sufficient to elicit the individual's immune response. The present invention relates to a method for inducing an immune response. Vaccines comprising a 5-helix (or variant or part thereof) in a physiologically acceptable carrier are the subject of the present invention.
[0009]
A 6-helix protein comprising three N-helices and three C-helices of HIV gp41 linked by a linker, such as an amino acid residue linker, is also the subject of the present invention. In one embodiment, the 6-helix protein comprises the amino acid sequence of SEQ ID NO: 2. In other embodiments, the 6-helix amino acid sequence differs from the sequence of SEQ ID NO: 2 by the addition, deletion, substitution, or modification of at least one amino acid residue. The 6-helix protein is useful as a negative control in screening for drugs that inhibit membrane fusion as well as 5-helix production.
[0010]
The submission of this patent contains at least one color drawing. Copies of this patent with this color drawing are made at the request of the Patent and Trademark Office and paid for.
[0011]
Detailed Description of the Invention
The higher order structure of the major part of the ectodomain of the gp41 molecule consists of a hairpin trimer structure. The central “hairpin trimer” is composed of a central triple-stranded N-helix coiled coil surrounded by three outer C-helices, which forms a bundle with all six helices. Hairpin trimers are common structural elements associated with the fusion of multiple enveloped viruses, suggesting an important role for this motif in promoting membrane fusion. In HIV gp41, the center of the hairpin trimer is centered on six alpha helices (of three gp41 ectodomains) encapsulated in an antiparallel fashion to the central triple-stranded coiled coil (formed by the N-terminal region of the gp41 molecule). (Formed by the C-terminal region) (M. Lu. Et al., J. Mol. Biol. 290, 1031-1044 (1995); DC Chan et al., Cell 89, 263-273 (1997); Weissenhorn et al., Nature 387, 426-430 (1997); K. Tan et al., Proc. Natl. Acad. Sci. USA 94, 12303-12308 (1997)). The hairpin trimer motif directs the two membranes together because the fusion peptide region inserted into the cell membrane is at the extreme N-terminus of gp41 and the C-terminal region is adjacent to the transmembrane helix anchored to the viral membrane. Useful for. This is illustrated in FIG. 1A. The N-helix (one of each subunit of the trimer) forms a very conserved hydrophobic groove encapsulating the C-helix. This is generally consistent with the formation of this 6-helix structure required to produce membrane fusion.
[0012]
The importance of hairpin trimer formation in HIV-1 entry sets the hypothesis that the C-terminal region of gp41 serves as a target for potential membrane fusion inhibitors. C-peptide is an in vitro IC₅₀It has been shown to inhibit HIV-1 entry into cells with values as low as 1 nM (CT Wild et al., Proc. Natl. Acad. Sci. USA 91, 9770-9774 (1994). DC Chan et al., Proc. Natl. Acad. Sci. USA, 95, 15613-15617 (1998)). This proof suggests that the C-peptide acts in a dominant negative manner by binding to the N-peptide region and disrupting hairpin trimerization. If the C-terminal region is accessible (at least transiently) prior to hairpin trimer formation, it is reasonable to expect that an agent that binds to this region at the N-terminus of gp41 will prevent membrane fusion. Consistent with this notion, peptides derived from the N-terminal region of gp41 (referred to as N-peptides) are suitable inhibitors of HIV-1 membrane fusion. The inhibition mechanism of N-peptides has not been determined, partly because these peptides tend to aggregate strongly.
[0013]
Applicants have found that one soluble molecule containing two of the C-helices of the folded N-helix center and the central hairpin trimer is very stable and one C-peptide with high affinity. And decided to combine. As described herein, the hypothesis that the C-peptide region of gp41 is a target for HIV-1 entry inhibition was examined. As also described herein, the results of the evaluation are strong for the HIV-1 variant in which the 5-helix that binds to the C-peptide region of gp41 is against HIV-1 and includes a set of different envelope proteins. It was clarified to show inhibitory activity. These results refer to the C-peptide region of HIV gp41 as a viable target for inhibiting the formation of hairpin trimers necessary for membrane fusion (and thus HIV infection of cells).
[0014]
Results are shown herein that show that proteins that bind to the C-peptide region of gp41 inhibit HIV entry into cells. Such proteins are inhibitors of HIV and serve as the basis for the development of additional anti-HIV drugs. These can be used to generate neutralizing antibody responses that target the N-terminal region of the gp41 ectodomain.
[0015]
A 5-helix, when indicated as a protein, takes advantage of the binding properties of the N-helix peptide coiled coil while minimizing the tendency of the N-peptide to aggregate. In one 5-helix embodiment, five of the six helices that comprise the center of the gp41 hairpin trimer structure are linked (coupled) to a short peptide linker (see FIG. 1A). In this embodiment, the 5-helix lacks a third C-peptide helix, thus providing a gap for obtaining a high affinity binding site for the C-terminal region of gp41. In a further embodiment of a 5-helix, three N-peptide helices and more than two (but less than three complete) C-peptide helices are connected by a short peptide linker. In these embodiments, three N-peptide helices, two complete C-peptide helices, and a portion of a third C-peptide helix are joined by a peptide linker. The portion of the third C-helix can be one amino acid residue of the third C-helix or any number of additional amino acid residues of the helix, but includes all amino acid residues of the helix Absent. The 5-helix proteins of the present invention are soluble under physiological conditions.
[0016]
The center of the hairpin trimer formed by individual N- and C-peptides is already very stable and the melting temperature is 65 ° C. Applicants have shown that when five of the six helices are covalently linked to form a 5-helix protein, the stability of the center is further increased (stability is higher than the stability of the 6-helix center). Indicated. Under physiological conditions, the 5-helix is folded, water-soluble and stable. This has an alpha helix content that closely matches the value expected from the design (see FIGS. 2A and 2B). In affinity interaction experiments, the 5-helix interacts strongly and specifically with the epitope-tagged C-peptide (see Figure 2C). As judged by the difference in circular dichroism before and after mixing, this interaction involves helical conformation in the bound C-peptide (see FIG. 2D). These properties are consistent with the intended design of the 5-helix.
[0017]
The 5-helix potently inhibits HIV-1 membrane fusion as measured by viral infectivity and cell-cell fusion assays (nanomol IC₅₀(See FIGS. 3A and 3B). In contrast, a control protein called a 6-helix occupied by a C-peptide bound by a C-peptide binding site (ie, all six of the gp41 hairpin trimer as described in Example 1). Of the helix bound to one polypeptide) does not show a clear inhibitory activity. (See FIG. 3A and FIGS. 8 and 9). Similarly, a 5-helix variant (referred to as 5-helix (D4)) in which the C-peptide binding site is disrupted by Asp mutations at four border residues (V549, L556, Q563, and V570) is 1 μM. But it does not block membrane fusion (see Example 3 and FIG. 3A). These results support the conclusion that the C-peptide bond is a key determinant of the antiviral activity of the 5-helix.
[0018]
The inhibitory activity of the 5-helix and C-peptide is expected to be antagonistic: when the 5-helix binds to the C-peptide, the amino acid residues thought to be responsible for the antiviral activity of each inhibitor are at the binding boundary. Buried. In fact, a mixture of 5-helix and C34 [well-characterized, IC₅₀Is a potent peptide inhibitor of about 1 nM] shows a dose-dependent antagonistic effect (FIG. 3B). In the presence of a 5-helix, inhibition of high titers by C34 is only observed when the peptide is in stoichiometric excess (Figure 3B).
[0019]
The 5-helix inhibits infection by viruses pseudotyped with various HIV-1 envelope proteins (clades A, B, and D) with similar titers (FIG. 3D). This widespread inhibition appears to affect the highly conserved boundary between the N-terminal region and the C-terminal region within the gp41 hairpin trimer structure (FIG. 4). Residues in the C-peptide region of gp41 that are predicted to contact the 5-helix are highly conserved in HIV-1, HIV-2, and SIV (FIG. 4).
[0020]
A 5-helix, a potent and broad inhibitor of viral entry, can serve as the basis for the development of a new class of therapeutics against HIV-1. Despite typically requiring parenteral administration, protein-based therapeutics such as insulin, growth hormone, tissue plasminogen activator, granulocyte colony stimulating factor, and erythropoietin may be practical. Alternatively, the 5-helix can be expressed endogenously (by gene therapy) and secreted into the bloodstream. If the 5-helix is endogenously expressed in HIV infected cells, it can inhibit intracellular folding and transport of gp160. 5-helix, 5-helix (D4), and 6-helix are also potential reagents for the purpose of screening small molecule drugs. The 5-helix has many degrees of freedom in the design of variants with better therapeutic properties. In principle, the 5-helix other than the C-peptide binding site can be extensively modified to change its immunogenicity, antigenicity, bioavailability, or inhibitory properties. For example, the C-peptide binding site in the gp41 sequence can be extended, shortened, or altered to optimize the inhibitory titer by targeting different regions of the gp41 ectodomain.
[0021]
It is desirable to produce neutralizing antibodies that mimic the binding properties of a 5-helix. The broad neutralizing ability of the 5-helix is likely derived from interaction with highly conserved residues in the C-peptide region of gp41 (FIG. 4). Since the linear sequence of the C-peptide region of gp41 can vary between different HIV-1 strains, an inconsistent C-peptide immunogen may not elicit extensively neutralizing antibodies. Such inconsistent C-peptides do not have long regions of conserved amino acid residues. Rather, conserved amino acid residues and non-conserved residues are interspersed. However, limiting a C-peptide or C-peptide analog to a helix structure (eg, in the C-peptide region when binding to a 5-helix) is useful as an immunogen in an effort to develop an AIDS vaccine. Can be. FIG. 4 is a helix wheel diagram showing the interaction between the C-peptide region of gp41 and the 5-helix. As shown, the entire “surface” on the helix wheel is made up of conserved or identical amino acid residues. Moreover, as shown, the degree of conservation of the positions a and d in the C-peptide region of HIV gp41 is high. From the C-terminal region of the gp41 ectodomain restricted in such a way that there are highly conserved amino acid residues (such as positions a, d, and e in FIG. 4) on one surface of the molecule Peptides can be produced. Use these peptides as immunogens to create antibodies that probably bind to these amino acid residues in the corresponding unrestricted peptide (C-peptide region of HIV gp41) and mimic the binding properties of a 5-helix. can do. For example, antibodies can be produced that bind to some or all of the C38 highly conserved (identical and / or conserved) amino acid residues (see FIG. 4). Such antibodies that mimic 5-helix binding actually act as prophylactics or vaccines by reducing or preventing 5-helix activity (binding). Such antibodies against restricted peptides derived from the C-terminal region of the HIV gp41 ectodomain are the subject of the present invention.
[0022]
Interestingly, the epitope of 2F5 (only known human monoclonal antibodies directed against gp41 with broad neutralizing activity) is adjacent to the C-terminus of the C-peptide region targeted by the 5-helix. Exists (T. Muster et al., J. Virol., 67, 6642-6647 (1993); M. Purtscher et al., AIDS 10, 5, 587-593 (1996)). The center of the 2F5 epitope (Leu-Asp-Lys-Trp; residues 663-666 in the HIV HXB2 gp160 sequence) is the HIV-1, HIV-2, and SIV isolate used to generate FIG. Are highly conserved in the same set of (81% identical). However, some HIV-1 escape variants to 2F5 neutralization do not contain mutations in the epitope sequence, suggesting that inhibition by 2F5 may be involved in recognition of additional determinants. Although the conformation of the 2F5-binding epitope remains unknown, antibodies raised with a fragment of gp41 containing this sequence have no significant virus neutralizing activity (T. Muster et al., J. Virol., 68, 4031-4034 (1994); L. Eckhart et al., J. Gen. Virol., 77, 2001-2008 (1996)). Whether 2F5 inhibits infection by interfering with hairpin trimer formation is unclear.
[0023]
Furthermore, the 5-helix itself is a vaccine candidate. It has been suggested that it may elicit an antibody response to the transiently exposed conformation of proteins associated with HIV-1 fusion (RA LaCasse et al., Science, 283, 357-362 (1999)). One possible well-defined target is the N-terminal coiled coil exposed to a pre-hairpin intermediate (DM Eckert et al., Cell, 99, 103-115 (1999)). A 5-helix-like intermediate can be exposed during the fusion process, in which case antibodies directed against the 5-helix can inhibit viral entry.
[0024]
The results described herein refer to the C-peptide region of HIV-1 gp41 as a viable target for inhibiting hairpin trimer formation. Structural and computational methods predict similar hairpin trimer motifs of viruses in many different families, including Orthomyxoviridae, Paramyxoviridae, Filoviridae, Retroviridae, and the like. Furthermore, in some of these cases, inhibition of virus entry by peptide analogs of the C-peptide of gp41 has been demonstrated. Thus, the 5-helix design approach can provide a widely applied strategy to inhibit viral infection.
[0025]
Furthermore, the 5-helix provides a means to study in detail the formed C-peptide binding site that cannot be done with aggregated N-peptides. The exposed C-peptide binding site in this 5-helix molecule is useful for identifying or designing molecules that bind to the N-helix center of gp41, and further using known methods such as HIV membranes and human cells The ability to inhibit the fusion of mammalian cells with the membrane and inhibit (reduce or prevent) cell infection can be assessed. Furthermore, the 5-helix can be evaluated for its ability to bind to the C-helix region of gp41 and inhibit its action. Since the N-helix center of gp41 is highly conserved (in terms of amino acid composition), the 5-helix and its variants appear to neutralize various HIV clinical strains extensively and are thus useful for therapy. .
[0026]
A 5-helix protein based on a known structure of the gp41 ectodomain, in one embodiment, consists of 3 N-peptides and 2 C-peptides covalently linked and 3 C-helix bonds occupied by C-peptides It is arranged to fold into a substantial part of the N-helix center with two of the sites. The remaining C-peptide binding sites of the N-peptide are exposed. This site exposes a 40 amino acid long epitope. Furthermore, since the N-peptide center is fixed by the two outer C-peptides, it is expected that the skeletal atom at this site is firmly held in the structured higher-order structure.
[0027]
The amino acid sequence of one embodiment of the 5-helix is shown below by one letter code for amino acids:

(SEQ ID NO: 1)
[0028]
The amino acid sequence of the 6-helix is shown below by the single letter code for amino acids:

(SEQ ID NO: 2)
[0029]
5-helix proteins can be made in a variety of ways. For example, as described in Example 1, it can be made from larger proteins (such as 6-helix) by enzymatic (trypsin) digestion. Alternatively, using known methods and expression systems, one DNA or two or more DNA “units” (each part of an N-helix protein (eg, one or Encoding a plurality of N-helices, one or more C-helices). Direct expression of the 5-helix gene in an appropriate host cell such as E. coli can significantly improve the expression rate and purification rate of the 5-helix. In this approach, the 5-helix gene encodes residues that are present in the final 5-helix protein. A C-terminal His tag can be attached to facilitate purification (with or without a protease cleavage site that subsequently removes the tag). The protein can then be used directly without the cleavage and folding steps that result in the proteolysis required to make the 5-helix starting from the 6-helix. This 5-helix molecule can be expressed as a folded active molecule, which can be used in biological selection or screening to optimize its properties. Alternatively, protein synthesis methods can be used to make 5-helix proteins. The five helices of the 5-helix are covalently linked (such as by linker means or other means of at least one (one or more) amino acid residues) and are stable under physiological conditions, A correctly folded protein can be formed so that the remaining surface is available for C34 peptide binding. In embodiments where there are three N-helices and more than two (but less than three full) C-helices, the helices can be similarly coupled.
[0030]
A 5-helix can have a wide variety of sequences in both the N- and C-helix regions as well as the linker component, which can be L-amino acid residues, D-amino acid residues, or L- and D- It can be composed of a combination of amino acid residues. Amino acid residues can be altered. The 5-helix may contain amino acid residues in addition to the helix and linker residues (eg, for molecular stabilization). It appears that the 5-helix described herein can be altered to improve stability and activity. Small changes in the design of the loops (both length and composition) linked to the N- and C-helices and the exact boundaries of the N- and C-helices are significant for 5-helix stability, yield, and activity Seems to have an effect.
[0031]
Similar to what is currently constructed, the 5-helix exposes a C-peptide binding site surrounded by 40 amino acids along the N-helix center. A strategy that exposes a shorter segment of the C-peptide binding site on the 5-helix (or related molecule) involves binding a short C-peptide sequence to a longer exposed epitope. This type of molecule can help develop drugs that are specifically targeted to shorter epitopes along the N-helix center. For example, one pocket region (similar to that found in IQN17; DM Eckert et al., Cell, 99, 103-115 (1999)) is bound there (about the first approximately 10 residues of C34). It can be exposed in a 5-helix by binding to a C-peptide lacking residues. These short C-peptide sequences can be attached to the 5-helix by various means including covalent cross-linking or simply extending the 5-helix sequence to cover a portion of the exposed epitope.
[0032]
5-helixes are useful in a variety of situations. As described herein, 5-helices are potent inhibitors of viral membrane fusion, so before the virus enters cells that act on HIV-infected cells (unlike current practical therapies) Acts on viruses. The 5-helix was found to be soluble and stable under the conditions described herein. It should also be possible to create 5-helix variants with increased molecular weight (by oligomerization or bundling large proteins) to reduce the clearance rate in the kidney. In addition, 5-helix dimers can be made by disulfide bridges to create molecules filtered to a smaller extent than 5-helix “monomers”. Thus, it is reasonable to expect that the dimer has improved bioavailability when compared to that of the C-peptide.
[0033]
5-helices, unlike standard therapies that target viral proteins after viral entry, prevent viral entry into cells, so 5-helix can be used prophylactically to prevent or prevent infection. The resulting range can be reduced. One application of such treatment is in situations where needlestick damage or HIV-contaminated needles, such as can occur in hospitals, are shared. For example, a needle can be inserted to prevent or reduce HIV invasion into cells, and a sufficient amount of 5-helix (therapeutically effective amount) can be applied one or more times to an individual who is or may be infected with HIV. Can be administered. The 5-helix can be administered, for example, by intravenous or intramuscular injection.
[0034]
In one embodiment of the invention, a 5-helix is used to reduce an individual's HIV infection. In this embodiment, expression of the 5-helix-encoding DNA in the 5-helix itself or in a suitable host cell or vector in an amount sufficient to reduce (in whole or in part) HIV infection of the individual's cells. The 5-helix is administered to the individual either via. That is, a dose (effective dose) of a 5-helix sufficient to reduce HIV infection, in a manner that inhibits (in whole or in part) HIV entry into the cell (eg, injection, local administration, intravenous route) ). In one embodiment, an effective dose is obtained using a gene therapy approach by introduction of cells expressing a 5-helix protein into an individual. The 5-helix can be administered to an HIV infected individual to reduce further infection or to an uninfected individual to prevent infection or reduce the extent to which infection occurs.
[0035]
Pharmaceutical compositions comprising a 5-helix in a suitable carrier (eg, a physiologically acceptable buffer) are the subject of the present invention. The pharmaceutical compositions are useful for prophylactic and therapeutic purposes and can be administered via various routes (eg, injection, topical administration, intravenous route).
[0036]
A 5-helix represents one intact C-helix binding site and is useful for screening drugs that inhibit membrane fusion. The 5-helix exposes a larger and more robust potential drug screening target than IQN17 exposes. This molecule 6-helix and 5-helix (D4) are useful negative controls in these studies.
[0037]
A 5-helix exposed epitope can be used as an antigen for the production of antibodies, particularly neutralizing antibodies using known methods. The antibody can be monoclonal or polyclonal.
[0038]
Serum stability of the 5-helix can be tested using known methods to confirm its therapeutic potential. When the 5-helix is degraded, the most promising attack / degradation site is the glycine / serine linker region. In this case, different linker regions can be degraded and tested (see below). The ability of these anti-5-helices to inhibit serum and ascites can be tested using standard fusion assays.
[0039]
The outer surface of the 5-helix can be altered, for example, to improve bioavailability, reduce toxicity, and avoid immune clearance. Since the 5-helix shows strong inhibitory activity but not the 6-helix bundle, this is an exposed groove containing the pocket region responsible for inhibition. The remaining molecules simply provide a scaffold for displaying exposed grooves. Thus, this scaffold can be modified without adversely affecting the inhibitory activity of the 5-helix. Several advantages can be obtained by modifying the scaffold. First, it facilitates procedures that require multiple administrations of 5-helix. For example, when using 5-helix as an anti-HIV therapeutic, multiple doses may be required. After prolonged administration, the individual can produce antibodies to the 5-helix that are thought to increase its clearance from the body. The usefulness of multiple versions of the 5-helix helps to avoid this problem by avoiding existing antibodies. Second, it may be possible to design a 5-helix version, for example, by introducing glycosylation sites on the outer surface where the scaffold is less immunogenic. For vaccine studies, this modification helps to deflect the immune response to the exposed groove as opposed to the scaffold.
[0040]
The finding that binding of the gp41 C-helix region prevents HIV infection suggests a strategy for constructing an HIV vaccine. The 5-helix, an HIV inhibitory analog by C-peptide, appears to inhibit gp41 by binding to a fusion intermediate of gp41 called a pre-hairpin intermediate. The C-peptide inhibitor appears to function by binding to the N-peptide region of this intermediate, while the 5-helix appears to function by binding to the C-peptide region. These considerations strongly suggest that the C-peptide region of gp41 is a good drug target for HIV entry inhibitor development. Furthermore, it is possible to use C-peptide based constructs as immunogens for raising neutralizing antibodies. In the case of a 5-helix, the target of inhibition is the helical conformation of the C-peptide region, but reagents that target other conformations of the C-peptide region may also exhibit inhibitory activity.
[0041]
Recent vaccine research (RA LaCasse et al., Science, 283, 357-362 (1999)) suggests that intermediates in the envelope-mediated fusion process can strongly elicit neutralizing antibodies. Antibodies against such fusion intermediates appear to target the conserved region of the envelope protein and thus neutralize a broad range of virus strains. Antibodies against the C-peptide region target regions that are highly conserved and important for the fusion process.
[0042]
Hairpin trimers are a common feature of many viral membrane fusion proteins. This has been observed in the crystal structures of influenza, Ebola, SV5 (simian parainfluenza virus 5), and RSV (human respiratory syncytial virus). In addition, many other members of the retrovirus, paramyxovirus, and filovirus families are expected to contain this motif. Similar structures have been observed in related vertebrate vesicle fusion proteins. The basic strategies described herein can be applied to any of these systems to inhibit fusion. One subject of the present invention is an envelope by contact of a virus with a drug that binds to a viral envelope protein (eg, the C-peptide region of the viral envelope protein) and inhibits heparin trimerization of the enveloped protein. It is a method for inhibiting the formation of hairpin trimers of viruses having viruses (viruses containing virus envelope proteins).
[0043]
The invention is illustrated by the following examples, which are in no way intended to limit the invention.
[0044]
Example 1: 5-Helix Production
The 5-helix design was based on the N36 / C34 6-helix bundle structure (DC Chan et al., Cell 89, 263-273, 1997). For a 5-helix, each peptide region is extended by three residues on its N-terminus and one residue on its C-terminus (compared to N36 and C34), creating the final N40 and C38 segments (Representative residues 543-582 and 625-662 of HIV-1 HXB2 gp160, respectively). Three N40 and two C38 segments were ligated using a -GGSGG-linker behind N40 and a -GSSGGG-linker behind C38. All constructs contain an N-terminal Met for translation initiation. The following two different 5-helix proteins that differ only in the C-terminus were generated for this study: (i) His-tagged 5-helix ending with GG (H) 6 and (ii) untagged ending with -GGR. Chemical 5-helix. In addition, a third construct called 6-helix was created in which the 5-helix backbone was linked to the His-tagged C-peptide (C37-H6) (see Example 4) by a trypsin cleavage linker (-GGR-). (See FIGS. 10 and 11A-11C).
[0045]
All DNA constructs were transformed into pAED4 vector [D. E. coli using E. coli XL1-Blue (recA strain, Stratagene). S. Doering, P.A. (Matsudaira, Biochemistry, 35, 12679-12585, 1996). All proteins were recombinantly expressed in E. coli strain RP3098 grown in 2 × YT to an OD (590 nm) of 0.5-0.7 prior to induction with IPTG (0.4M) for 3 hours. Bacterial pellets were resuspended in Tris / NaCl buffer (Qiaexpressionist booklet, March 1999, Qiagen) supplemented with complete EDTA-free protease inhibitor tablets (Roche) and then frozen at −20 ° C. until the day of purification. Thawed resuspension was lysed (sonication or French press) and centrifuged (35,000 × g for 30 minutes) to separate the soluble fraction from the inclusion bodies.
[0046]
His-tagged 5-helix (made from plasmid p-5HelixH6) was purified directly from inclusion bodies resuspended in 8M urea TBS (50 mM Tris (pH 8.0), 100 mM NaCl) and 10 mM imidazole solution. The mixture was clarified by centrifugation (35,000 × g for 30 minutes) and then bound to a Ni-NTA agarose (Qiagen) column at room temperature. The protein was eluted with 40 ml (about 5 column volumes) of 6M urea / TBS / 100 mM imidazole solution. The protein was refolded by dropwise addition of 1 liter of 20 mM Tris (pH 8.0) stirring solution at room temperature. The refolded protein was then reconcentrated by passage through a Ni-NTA agarose column and eluted with 20 ml (approximately 2 column volumes) of 100 mM imidazole in TBS.
[0047]
Untagged 5-helix was produced by proteolysis of 6-helix (see below), creating a 5-helix / C37-H6 complex. After trypsin digestion (1: 200 weight ratio in TBS, 1 hour at room temperature, Sigma), the 5-helix / C37-H6 complex was bound to Ni-NTA agarose and washed extensively to remove excess trypsin. Removed. The beads were resuspended in 8M GuHCl / TBS and heated (70 ° C.) to denature the complex. Unbound fractions containing the denatured 5-helix were serialized with 8M urea / 20 mM Tris (pH 8.0) (4 hours at room temperature) and 4M urea / 20 mM Tris (pH 8.0) (overnight at 4 ° C.). Dialyzed. The protein was loaded onto a DEAE column (Fastflow, Pharmacia) and run on a reverse urea gradient (4 mM to 0 M urea in 20 mM Tris pH 8.0) at room temperature for 4 hours at room temperature. The protein was eluted from the DEAE resin using a NaCl gradient (0-300 mM) in 20 mM Tris solution (pH 8.0) (10 column volumes). The 6-helix (made from plasmid p-6 helix) was purified directly from the soluble fraction of the bacterial lysate. The solution was passed through a Ni-NTA agarose column and eluted with 10 column volumes of imidazole gradient (10-250 mM) in TBS.
[0048]
For all proteins, monomers were separated from aggregates by gel filtration in TBS (Sephaacryl S200HR or Superdex 75). The protein is more than 95% pure as judged by SDS-PAGE and can be concentrated to at least 3 mg / ml. All peptide and protein concentrations were identified by absorbance at 280 nm in 6MGuHCl [H. Edilhoch, Biochemistry 6, 1948-1954, (1967). ]
[0049]
Example 2: Evaluation of specificity of 5-helix / C-peptide interaction and inhibition of membrane fusion by 5-helix
Specificity of 5-helix / C-peptide interaction was tested using His-tagged C-peptide (C37-H6 independently expressed in E. coli and purified by reverse phase HPLC) and Ni-agarose precipitation. did. In TBS containing 30 μM C37-H6, 16 μM 5-helix is completely precipitated by Ni-agarose. Addition of 150 μM C34 (without His tag, chemically synthesized and purified by HPLC) substantially reduces the amount of 5-helix precipitated. Effective competition between C37-H6 and C34 indicates that the 5-helix binds to the C-peptide in a specific manner. CD experiments and competitive binding assays suggest that the 5-helix is folded into the predicted conformation. That is, these results support the expectation that the 5-helix contains an exposed C-peptide binding site.
[0050]
An assay was performed to assess the ability of the 5-helix to interact with the C region of gp41 to inhibit fusion protein fusion. Inhibition of this membrane fusion by 5-helix and 6-helix was assessed using a cell-based assay. Proteins 5-helix and 6-helix were serially diluted in modified DMEM medium containing 5% FCS and aliquoted into slide chambers. HELA cells expressing CD4 and co-receptors under the control of the Tat promoter and containing the β-galactosidase gene (4 × 10 4^Four ) Is added. CHO cells expressing gp160 (a precursor protein of gp120 / gp41) and Tat (2 × 10^Four ) Is also added. 400 μl of mini-medium is incubated at 37 ° C. for 8-24 hours, and the fused cells (syncytium) transcribe and translate β-galactosidase. Cells are fixed in glutaraldehyde and exposed to X-gal / Fe solution for 1 hour. Syncytium containing β-galactosidase turns blue-green. In this assay, the 5-helix has shown potent inhibition of syncytium formation (IC₅₀10-20 nM; in some assays, 13 nM IC₅₀Met). The 6-helix does not appreciably interfere with fusion even at a concentration of 1 μM.
[0051]
  To evaluate the specificity of the 5-helix exposed epitope as an inhibitor and to exclude contaminants, mixing experiments with C-peptide were performed. A concentration of 200 nM 5-helix is mixed with 100, 166, 190, and 210 nM C34. At the concentrations used, free 5-helix and free C34 should inhibit almost all syncytium in the miniculture. In the 5-helix / C34 mixture where C34 is in excess of the 5-helix (ie 210 nM), syncytium formation isBeing disturbed, Syncytium formation in 5-helix / C34 mixtures where the C34 concentration is lower than the 5-helix concentration isPartiallyUnimpeded(Fig. 3B). In contrast, C34 blocks all syncytium formation in the presence of a 6-helix.
[0052]
The inhibitory potential of 5-helix and 6-helix was reproduced in virus fusion experiments. HIV modified to contain a luciferase reporter gene is mixed with human osteosarcoma (HOS) cells expressing CD34 and co-receptors for 6 hours at 37 ° C. in the presence of diluted protein. The virus solution is changed and the HOS culture is incubated in fresh medium for at least 48 hours. Luciferase activity is measured with a luminometer. In this assay, the 5-helix has an IC less than 10 nM.₅₀Inhibits luciferase activity. Also, the 6-helix does not show appreciable interference up to 1 μM (FIGS. 3A and 3C).
[0053]
Example 3: Design and evaluation of 5-helix (D4)
In 5-helix (D4), the 4 highly conserved residues (Val549, Leu556, Gln563, and Val570) in the His-tagged 5-helix C-peptide binding site are the last (third) Mutated to Asp in the N40 segment. The construct [p-5Helix (D4)] was recombinantly expressed and purified in the same manner as the His-tagged 5-helix. His-tagged 5-helix and 5-helix (D4) proteins have the same ellipticity: (both [θ] in TBS at 4 ° C.₂₂₂ = -28,100 ± 1500 deg cm²   dmol^-1(Estimated helix content of about 100%)), both proteins are heat denatured in TBS (Tm above 98 ° C.) and GuHCl chemical denaturation at 25 ° C. (Cm value is about 6 M in 5-helix (D4)) , His-tagged 5-helix to about 7.2M). The slightly reduced stability of the 5-helix (D4) is due to the low helix tendency of the Asp residues that are mostly in the hydrophobic groove on the surface of the 5-helix in this situation. And it seems to reflect the effect on the charge.
[0054]
Example 4: His-tagged C-peptide C37-H6
Peptide C37-H6 is a His-tagged C peptide with the following sequence:
GHHTTWMEWDREINNYTSLIHSLIIESQNQQEKNEQELLGHHHHHH (SEQ ID NO: 5). This peptide is derived from HIV-1 HXB2 residues 625-661 (underlined) and contains the entire C34 sequence (W628-L661). C37-H6 is made from a trypsin digest of the recombinant expression construct p4-NC1.1 consisting of one N40 segment joined to C37-H6 with a -GGR-linker. After expression, NC1.1 is purified from the soluble fraction of bacterial lysate in the same manner as the 6-helix. Trypsin digestion (same conditions as untagged 5-helix) yielded C37-H6, then reverse phase using a Vydac C-18 column and a linear gradient of aqueous acetonitrile containing 0.1% trifluoroacetic acid. Purify homogeneously by HPLC. The identity of C37-H6 was confirmed by mass spectrometry (MALDI-TOF, PerSeptive). Similar to C34, C37-H6 is a potent inhibitor of HIV-1 membrane fusion (IC in cell-cell fusion assays).₅₀Is about 1 nM).
[0055]
The data in FIGS. 2A-2D were generated using a 5-helix untagged variant, but similar results were obtained with the His-tagged variant (see Example 3). Unless otherwise stated, CD (Aviv62DS) experiments were performed in TBS buffer. In FIG. 2B, the protein concentration was 1 mM for the TBS sample and 0.54 mM for the GuHCl / TBS sample. In FIG. 2D, a quartz mixing cell (Helma) with a 1 ml chamber (4.375 mm / chamber optical path length) was used. The polypeptide concentration in 20 mM Tris (pH 8.0) / 250 mM NaCl before mixing was 5.9 mM (5-helix) and 6 mM (C37-H6).
[0056]
A 5-helix precipitation experiment (FIG. 2C) was performed in 20 ml TBS containing 16 mM untagged 5-helix, 30 mM His-tagged C37-H6, and / or 150 mM C34. The solution was added to Ni-NTA and incubated for 10 minutes at room temperature. After removing unbound supernatant, the beads were washed twice with 1 ml TBS and eluted with 500 mM imidazole. Ni bound and unbound samples were run on a 16.5% Tris-Tricine polyacrylamide gel (Biorad) and stained with Gel-code Blue (Pierce).
[0057]
Example 5: His-tagged 5-helix
All data in FIGS. 3A-3C were generated using a His-tagged 5-helix (see Example 1). A cell-cell fusion assay (FIG. 3B) was performed as described (DC Chan et al., Proc. Natl. Acad. Sci. USA 95, 15613-15617, 1998). Inhibition of viral infection using a recombinant luciferase reporter assay with a slight modification of a previously detailed assay (DC Chan et al., Proc. Natl. Acad. Sci. USA, 95, 15613-15617, 1998). Studied. Briefly, the envelope-deleted HIV-1 genome NL43LucR^- E^- [B. K. Chen et al. Viol. , 68, 654-660, 1994] and one of the following four gp160 expression vectors: pCMV-HXB2 (DC Chan et al., Proc. Natl. Acad. Sci. USA, 95, 15613-15617, 1998), pEBB-JRFL (provided by BK Chen), pSVIII-UG024.2, and pSVIII-RW020.5 co-transfected 293T cells to generate pseudotyped viruses. Plasmids pSVIII-UG024.2 and pSVIII-RW020.5 were obtained from the NIH AIDS reagent program (F. Gao, B. Hahn, and DAIDS, NIAID), which produced the envelope protein from the primary HIV-1 isolate. Code. Supernatant containing virus was prepared as previously described (DC Chan et al., Proc. Natl. Acad. Sci. USA 95, 15613-15617, 1998) and used to produce HOS-CD4 cells (HXB2 And UG024.2) or HOS-CD4-CCR5 cells (JRFL and RW020.5). Cells were obtained from the HIH AIDS reagent program (N. Landau). In FIG. 3A, a viral infectivity assay was performed in a standard 24-well format (DC Chan et al., Proc. Natl. Acad. Sci. USA 95, 15613-15617, 1998). The data in FIG. 3C was obtained from an assay performed in the following 96-well format: virus containing supernatant (10 ml) and medium (90 ml) were overlaid on 50% confluent HOS cells. After 2 days incubation at 37 ° C., cells were harvested in 100 ml lysis buffer (Luciferase Assay System, Promega) and 10 ml was analyzed according to the manufacturer's protocol. Langmuir function [standardized luciferase activity = 1 / (1+ [5-helix] / IC₅₀)] By fitting the 5-helix titration data to the IC₅₀The value was calculated.
[0058]
Although the invention has been shown and described with reference to particularly preferred embodiments, various changes in form and detail can be made without departing from the scope of the invention as encompassed by the appended claims. Will be understood by those skilled in the art.
[Brief description of the drawings]
1A and 1B show targeting of HIV-1 membrane fusion. FIG. 1A is a schematic representation of HIV-1 membrane fusion showing events that promote the formation of gp41 hairpin trimers. A gp41 (red) N-terminal fusion peptide that is inaccessible in nature is inserted into the target cell membrane after interaction of gp120 with CD4 and co-receptors (not shown). The structure of the pre-hairpin intermediate is exposed by the N-terminal coiled coil (grey) (target of C-peptide inhibition). This transient structure collapses into a hairpin trimer state, and the membranes fuse and are placed closely in parallel. FIG. 1B shows the design of the 5-helix construct. The ribbon diagram (top) shows the central structure of the hairpin trimer (left and DC Chan et al., Cell 89, 263-273 (1997)) and the 5-helix model (right). The inner gray helix represents the N36 peptide and the outer blue helix represents the C34 peptide. In the 5-helix model, one C-peptide has been removed and the orange line is drawn to show the connection between the helices. In the 5-helix design, short Gly / Ser peptide sequences (gray bars at the bottom of the figure) alternate N40 and C38 sequences (shown with single letter amino acid symbols) (see Example 1). .
FIGS. 2A-2D show the 5-helix nature. FIG. 2A is a circular dichroism (CD) spectrum of 5-helix (10 μM) at 25 ° C. This spectrum shows that the 5-helix protein uses a helix content greater than 95% expected from the design. FIG. 2B is a graph of 5-helix thermal denaturation monitored by ellipticity at 222 nm in TBS (black squares) and 3.7 M guanidine (Gu) HCl / TBS (white squares). The denaturation observed with GuHCl solution is more than 90% reversible. FIG. 2C shows the results of 5-helix nickel (Ni) -NTA precipitation with His-tagged C-peptide. Untagged 5-helix and His-tagged C-peptide (denoted C37-H6) were mixed before adding Ni-TNA agarose to precipitate the complex containing C37-H6 (lanes 1 and 5 and Example 4). Addition of excess untagged C-peptide (C34) changes the 5-helix molecule from bound to unbound fraction (lanes 2 and 6). FIG. 2D is a CD spectrum of 5-helix and C37-H6 before mixing (black squares) and after mixing (white circles) in a mixing cuvette. An increase in ellipticity at 222 nm due to mixing indicates an interaction between the two species that corresponds to an increase in overall helix content (corresponding to an additional 28 helix residues per associated C-peptide).
3A to 3C show the evaluation results of 5-helix inhibition of HIV-1 envelope mediated membrane fusion described in Example 2. FIG. FIG. 3A shows the results of titration evaluation of virus infectivity with 5-helix (black square), 6-helix (white triangle), and 5-helix (D4) (white circle) described in Examples 3 and 5. Data are shown as mean ± SEM of two or more individual experiments. FIG. 3B is a graph of the competitive inhibitory activity of 5-helix and C34. Syncytium counts were determined in cell-cell fusion assays performed in the presence or absence of the indicated concentrations of 5-helix, C34, or a mixture of 5-helix and C34. 5-helix and C34 IC in this assay₅₀The values are 13 ± 3 nM and 0.55 ± 0.03 nM, respectively (DC Chan et al., Proc. Natl. Acad. Sci. USA 95, 15613-15617 (1998)). The data shows the mean and range of duplicate measurements other than the control (mean of 5 measurements ± SEM). FIG. 3C shows the evaluation results of 5-helix inhibition of pseudotyped viruses containing different HIV-1 envelope glycoproteins. Reported IC₅₀Values are shown as mean ± SEM of 3 independent experiments.
FIG. 4 is a helix wheel diagram showing the interaction between the 5-helix and the C-peptide region of gp41. The positions ag in each helix indicate consecutive positions in the 4,3-hydrophobic 7 residue repeat in each sequence. The positions of a and d in the gp41 C-peptide interact with the positions of e and g exposed on the 5-helical N40 coiled coil. Surrounding residues according to the degree of conservation determined from alignment of 247 sequences from HIV-1, HIV-2, and SIV isolates (HIV-1 sequence database, August 2000, Los Alamos National Laboratory): black Squares (> 90% identical), gray squares (> 90% conservative substitution), dotted squares (70-90% conserved), no box (<70% conserved). In the creation of FIG. 4, substitutions within the following amino acid residue groups were considered conservative substitutions: [Asp, Glu], [Lys, Arg], [Asn, Gln], [Phe, Tyr], [Ser, Thr], and [Val, Ile, Leu, Met]. The high degree of conservation at positions a and d in the C-peptide region of gp41, a characteristic that is notably prominent at other positions in the C-peptide region (especially c and g), is directly involved in 5-helix binding. Note that you don't.
FIG. 5 is an organization of the structure of HIV gp41. The helix region (7 residue repeat) is shown in gray and the relative position of the N- (N36) and C- (C34, DP178) peptides. In the ribbon diagram of the helix region, the N-peptide is shown in light gray and the C-peptide is shown in dark gray.
FIG. 6 is a 6-helix and 5-helix sequence. Putative helix segments are indicated by sequence stacking.
FIG. 7 is a ribbon diagram of one possible α-helix organization of a 5-helix. The N-helix trimer is light gray, the C-helix region is dark gray, and the extension loop region is black (in the structure of DC Chan et al. (Cell 89, 263-273 (1997)). Based).
FIG. 8 shows an image of a cell-cell fusion assay titration experiment. In the image, syncytium (indicating fused cells) is blue and debris is brown.
FIG. 9 shows an image of a cell-cell fusion assay competition experiment. Record the amount of syncytium for cultures incubated in 200 nM 6-helix or 5-helix with increasing amounts of C34 peptide.
FIG. 10 is a diagram showing the design of a 5-helix construct. This schematic shows the binding pattern of the basic 5-helix construct. Three different C-termini were added. In 6-helix, a His-tagged C-peptide is attached to the 5-helix to mimic the complete 6-helix bundle of the hairpin trimer. N40 and C38 sequences (alternately linked using short Gly / Ser linkers) are derived from the N- and C-peptide regions of HIV HXB2 gp41. (The residues enclosed in red and blue indicate the peptide sequences of N36 (SEQ ID NO: 11) and C34 (SEQ ID NO: 12), respectively).
11A to 11C show the amino acid sequences of the peptides of the present invention (SEQ ID NOs: 1 to 10).

Claims (22)

  A C-peptide of HIV gp41, which is soluble under physiological conditions and comprises three N-helices of a trimer of the hairpin structure of HIV gp41 and at least two but not completely three C-helices A 5-helix protein that binds to a region, wherein the helices are separated by a linker.   The 5-helix protein of claim 1, wherein the linker comprises at least one amino acid residue.   5-helix protein comprising SEQ ID NO: 1. A method of identifying a candidate inhibitor of H IV infection, the method comprising,
Combining a compound or molecule with a 5-helix protein under conditions suitable for binding to occur, wherein the 5-helix protein comprises at least three N-helices of the trimer of the hairpin structure of HIV gp41. And two but not completely three C-helices and binds to the C-peptide region of HIV gp41, and
Determining whether binding occurs between the 5-helix protein and the compound or molecule, and if binding occurs, the compound or molecule is a candidate inhibitor of HIV infection . 5. The method of claim 4 , further comprising determining whether the compound or molecule that binds to the 5-helix protein inhibits HIV infection of mammalian cells in a cell-based assay.   Use of a composition comprising a 5-helix protein and a physiologically acceptable carrier in the manufacture of a medicament for inducing an immune response to HIV in an individual, wherein the 5-helix protein comprises HIV gp41 Uses comprising three N-helices and at least two but not completely three C-helices of a trimer of the hairpin structure of and binding to the C-peptide region of HIV gp41.   Binds to the C-peptide region of HIV gp41, inhibits HIV infection of mammalian cells, and at least two but not completely three C-trimers of the hairpin structure of HIV gp41 A 5-helix protein comprising a helix. 8. The 5-helix protein of claim 7 that inhibits HIV infection of human cells.   Prevents the formation of hairpin structure HIV gp41 trimer, inhibits HIV infection of cells, and at least two but not all three N-helices of hairpin structure trimer of HIV gp41 5-helix protein comprising C-helix.   Inhibits fusion of HIV and mammalian cell membranes, as measured by viral infectivity assays, cell-cell fusion assays, or both, and at least two of the three N-helices of the hairpin structure of HIV gp41 A 5-helix protein comprising a C-helix, but not all three. 11. The 5-helix protein of claim 10 , wherein the mammalian cell membrane is a human cell membrane. Viral infectivity assays or cell - as measured in the cell fusion assay, the protein inhibits HIV membrane fusion at nanomolar IC _50, claim 10 5- helix proteins according.   Use of a 5-helix protein that binds to the C-peptide region of HIV gp41 in the manufacture of a medicament for inducing an neutralizing anti-HIV response in an individual, wherein the 5-helix protein is of the hairpin structure of HIV gp41. Use comprising three N-helices of a trimer and C-helices of at least two but not completely three.   Use of a 5-helix protein that binds to the C-peptide region of HIV gp41 in the manufacture of a medicament for inhibiting cellular HIV infection in an individual, wherein the 5-helix protein comprises three of the hairpin structures of HIV gp41. Use comprising 3 N-helices of a mer and at least 2 but not completely 3 C-helices and binding to the C-peptide region of HIV gp41.   A 5-helix protein that inhibits the formation of the HIV gp41 hairpin trimer, or a neutralizing antibody that mimics the binding properties of a 5-helix protein, used to inhibit fusion of the HIV membrane with the human cell membrane in an individual The 5-helix protein comprises three N-helices of a trimer of the hairpin structure of HIV gp41 and a C-helix which is at least two but not completely three, and the HIV gp41 C A neutralizing antibody that mimics the binding properties of a 5-helix protein or 5-helix protein that binds to the peptide region. A 5-helix protein that binds to the HIV virus envelope protein or an antibody that mimics the binding properties of a 5-helix protein, used to inhibit the formation of a hairpin trimer of HIV gp41 , said 5-helix protein Contains 3 N-helices of the trimer of the hairpin structure of HIV gp41 and at least 2 but not completely 3 C-helices and binds to the C-peptide region of HIV gp41. An antibody that mimics the binding properties of a helix protein or a 5-helix protein.   A 5-helix complex comprising a 5-helix protein linked to a molecule that binds to an HIV envelope protein, wherein the 5-helix protein is a trimeric of hairpin structures of HIV gp41. And a 5-helix complex that binds to the C-peptide region of HIV gp41, and at least two but not completely three C-helices. 18. The 5-helix complex of claim 17 , wherein the molecule that binds to an HIV envelope protein binds to HIV gp120. 19. The 5-helix complex of claim 18 , wherein the molecule that binds to HIV gp120 is sCD4 or an antibody. Use of a 5-helix complex that binds to an HIV envelope protein in the manufacture of a medicament for inhibiting fusion of an HIV membrane and a human cell membrane in an individual, wherein the 5-helix complex binds to an HIV envelope protein A 5-helix protein linked to a molecule, wherein the 5-helix protein is a trimeric N-helix of the hairpin structure of HIV gp41 and a C-helix that is at least two but not completely three And binds to the C-peptide region of HIV gp41. 21. Use according to claim 20 , wherein the envelope protein is HIV gp120. (A) SEQ ID NO: 1, (b) SEQ ID NO: 7, (c) SEQ ID NO: 8, and (d) SEQ ID NO: An isolated protein selected from the group consisting of 9.

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